CN104855278A - Breeding method capable of reducing conversion rate of tobacco nicotine - Google Patents

Abstract

The invention relates to a breeding method for reducing the conversion rate of tobacco nicotine. The breeding method utilizes male sterile heterosis in the first generation, and the materials used include male sterile female parent, maintainer line and male parent. The male sterile line hybrid female parent The nicotine conversion rate of this and the maintainer and the male parent must be lower than 5%, and the nicotine conversion rate of the male sterile hybrid female parent and the maintainer must be lower than that of the male parent; the nicotine conversion rate is higher than 5%. % male sterile line hybrid female parent and maintainer line and male parent must be selected and purified for nicotine conversion rate; the male sterile line hybrid female parent nicotine conversion rate selection and purification with a nicotine conversion rate higher than 5% The purification includes simultaneous purification of the female parent of the male sterile line and the maintainer line. This method has found that the trait of nicotine conversion rate has negative heterosis in the process of hybridization, that is, the nicotine content of the hybrid F1 is greater than the average value of the parents, and the nornicotine is significantly lower than the average value of the parents, and the nicotine conversion rate is the lowest. When the parent is used as the female parent of the hybrid, the heterosis is the largest, the operation is simple, and the effect is remarkable.

Description

A breeding method for reducing the conversion rate of tobacco nicotine

technical field

The invention belongs to the field of tobacco breeding, and in particular relates to a breeding method suitable for utilizing male sterile first-generation heterosis for reducing the conversion rate of tobacco nicotine.

Background technique

Tobacco alkaloids mainly include nicotine, nornicotine and trace amounts of anonicotinoid and pseudobasine. Among them, nicotine is the main alkaloid of common tobacco varieties, with a content of more than 92%; nornicotine is mainly formed by demethylation of nicotine directly catalyzed by demethylase, so nornicotine is also called demethylnicotine, This demethylation ability of nicotine is the nicotine conversion ability. Under normal circumstances, the conversion of nicotine to nornicotine in common tobacco is controlled by the homozygous recessive genotype, which does not have the ability to demethylate nicotine, so the content of nornicotine generally does not exceed 3.5%. However, in the tobacco plant population of cultivars, some plants will develop nicotine conversion ability due to gene mutation, resulting in a significant decrease in nicotine content and a corresponding increase in nornicotine content. Compared with nicotine, nornicotine is more unstable, and it is easy to undergo biochemical transformation to form a series of nornicotine derivatives in the process of tobacco leaf preparation and aging, which seriously affects the quality and safety of tobacco leaves. For example, nornicotine undergoes an oxidation reaction to form mesminine, and an acylation reaction to form a series of acyl nornicotine such as formylnornicotine and acetylnornicotine, which makes the tobacco leaves emit an alkaline and rat odor. Not only that, nornicotine easily reacts with nitrous acid to form a strong carcinogenic substance - NNN (N-nitroso nornicotine). NNN is one of the four major tobacco-specific nitrosamines (TSNA) identified at present, and its content accounts for 80-90% of the total TSNA. As early as the 1950s, it was found that the flavor of "cherry red" tobacco leaves was not good, the nicotine content was greatly reduced, and the nornicotine content was significantly increased after the flue-cured tobacco was prepared. . In the 1980s, it was discovered that burley tobacco generally had nicotine conversion problems and that nicotine conversion was closely related to the formation of NNN, which attracted people's attention. Since the late 1990s, it has gradually become a research hotspot in the global tobacco technology community. In 1977, the U.S. industry established the maximum limit of three alkaloids, nornicotine, neonicotinoid and pseudobasine, in Burley tobacco leaves. The total proportion of the three alkaloids should not exceed 20%. Reduced to 15%, which is mainly nornicotine. At present, the main method to control the conversion of nicotine to nornicotine and reduce the content of nornicotine is to remove the mutants that undergo nicotine conversion in the original population of cultivars (i.e., the original species), so that the average nicotine conversion of production species rates remain low. For example, the promotion of burley tobacco production in the United States must be the low nicotine conversion rate variety marked with "LC". Our country started the research on nicotine conversion at the beginning of this century. Shi Hongzhi et al. concluded that the nornicotine content and nicotine conversion rate of burley tobacco were higher after studying the different types of tobacco in China and the conversion of nicotine to nornicotine. , the problem is prominent. After Li Jinping and others genetically improved the nicotine conversion of Burley tobacco Eyan 1, the nicotine conversion rate, nornicotine content and NNN content were all greatly reduced. Subsequently, Sichuan and Yunnan also successively published reports on the variety improvement of burley tobacco nicotine transformation. However, it is rare to see reports on the cultivation of low-nicotine transformation new varieties by breeding methods.

Tobacco breeding methods include: systematic breeding, sexual hybrid breeding, male sterile hybrid generation advantage utilization, mutation breeding, cell and genetic engineering breeding, etc. The first three are the most effective in breeding. Among them, the use of male sterile hybrids in the first generation is the main method for breeding new varieties of burley and flue-cured tobacco. The United States is the country that first applied this method and bred the most varieties. New varieties of flue-cured tobacco such as Yan 1-6, Eyan 209, 211 and other burley tobaccos, and Yunyan 97 and Yunyan 202. The first-generation dominant parents of male sterile hybrids include the female parent of the male sterile line, the maintainer line, and the male parent. Among them, the pistils and stamens of the flowers of the maintainer tobacco plants are fully developed and can self-fertilize; the pistils in the flower organs of the male sterile lines are well developed, but the stamens are degenerated. Progeny plants remain sterile. In the first-generation hybrid combination, the male sterile line is used as the female parent, and the seeds produced by crossing with a specific male parent, that is, the first-generation male sterile hybrid (hybrid F1 for short), are directly used in tobacco leaf production. Tobacco is a plant for leaves, and it does not need a hybrid plant to restore fertility and fruit, so there is no restorer line (different from food crops).

Nicotine is synthesized in the root of the tobacco plant, which is a quantitative trait, genetically controlled by 2 to 3 pairs of additive-effect micro-effect genes, and the hybrid F1 generation is close to the average value of the parents. Nornicotine is transformed from nicotine, which mainly occurs in the leaves, veins and stems of tobacco leaves during the curing period. The ability of nicotine to nornicotine transformation is genetically determined by 1 to 2 pairs of dominant genes, so nornicotine It is also dominant or partially dominant of nicotine, the hybrid F1 of nicotine type and nornicotine type, which mainly synthesizes nornicotine ("Tobacco Breeding", edited by Tong Daoru, China Agricultural Press, 1997, P145～146) . That is to say, it is impossible to breed varieties with low nicotine conversion rate and low nornicotine content by adopting the method of male sterile cross generation advantage utilization. At the same time, the nicotine conversion traits are different from the traits that can be directly judged by measuring tools or human senses, such as leaf size, color depth, yield, etc., and must be tested indoors after tobacco leaf modulation or after fresh leaves have been artificially induced. It is known that, in addition, nicotine conversion is caused by gene mutation, so there is greater uncertainty and instability in heredity, thereby increasing the difficulty of breeding.

Contents of the invention

According to the principle of first-generation advantage utilization of male sterile hybrids, the present invention has invented a method for reducing tobacco nicotine conversion through repeated research. The female parent and male parent of a specific male sterile line are selected for artificial hybridization to prepare a first-generation hybrid (F1) combination, and the resulting seeds are directly applied to tobacco leaf production, that is, hybrid F1 species or hybrids. The main principle is: use the hybrid advantage generated by gene dominant effect, epistasis effect, additive effect and gene modification effect after the combination of parents in the process of hybridization to meet people's needs for tobacco leaves in various aspects. The pros and cons of hybrids are usually measured by the size of heterosis and control vigor, and the calculation formula is: heterosis (%)=[(F1-average value of parents)/average value of parents]×100, a value equal to 0 means no heterosis, Greater than 0 indicates positive heterosis, and less than indicates negative heterosis; control vigor (%)=[(F1-control)/control]×100.

The invention provides a breeding method for reducing the conversion rate of tobacco nicotine, which adopts a method for utilizing male sterile hybrid vigor in the first generation, and the materials used include male sterile female parent, maintainer line and male parent, and the male sterile hybrid female parent And the nicotine conversion rate of the maintainer line and the male parent must be lower than 5%, and the nicotine conversion rate of the male sterile line hybrid female parent and the maintainer line must be smaller than the male parent; the nicotine conversion rate is higher than 5%. The male sterile line hybrid female parent, maintainer line and male parent must be selected and purified for nicotine conversion rate; the nicotine conversion rate selection and purification of the male sterile line hybrid female parent with a nicotine conversion rate higher than 5% Including the simultaneous purification of the female parent of the male sterile line and the maintainer line;

Specific steps are as follows:

1), the female parent of the male sterile line, the maintainer line and the hybrid male parent are each planted with 40-60 plants, fertilized and cultivated according to the conventional tobacco leaf production method;

2), when the tobacco plant is 30-40cm high and the number of leaves is 12-15 pieces, according to the number of the individual plant, collect 1-2 pieces of growth qualitative leaves of the 8th-10th leaf position from bottom to top, spray on the leaves after stringing Apply 0.3% ethephon to induce the conversion rate of nicotine, dry it in the air for 7-8 days with moisture in the room, and after the leaves turn yellow completely, dry them separately according to the number of each plant to make powder samples, and test the content of nicotine and nornicotine , to calculate the nicotine conversion rate;

Calculation formula: nicotine conversion rate (%)=[nornicotine/(nicotine+nornicotine)]×100; nicotine conversion rate <5% is non-conversion, 5-20% low conversion, 20-50% Medium conversion, >50% is high conversion;

3) According to the detection results of the transformation rate, find the corresponding numbers of individual plants in the field, keep 5-10 individual plants with relatively low transformation rates, and top or pull out other higher transformed plants.

4) The individual plants selected in the maintainer line and the male parent population are bagged and self-bred to save seeds, and the seeds are mixed and collected after harvesting; the pollen of the selected maintainer individual plants is cross-pollinated with the non-transformed strains in the sterile line population.

5) After the seeds of the selected individual plants are mature, they are harvested according to the individual plant number, mixed according to the variety after threshing, the variety name is marked, and the next generation is selected for use in the next year.

The selection and purification of the conversion rate of nicotine need to be repeated for more than 3-4 generations until the plants of the sterile line, the maintainer line and the male parent population are completely non-transformed plants with a conversion rate of <5%. Except for fertility, the nicotine conversion rate and other traits are exactly the same, so that the hybrid F1 combination can be prepared.

Main advantage of the present invention:

The present invention provides a breeding method for reducing the conversion rate of tobacco nicotine according to the principle of utilizing the superiority of the first generation of tobacco male sterile hybridization. (TSNAs) content is relatively high, which greatly improves the safety and comprehensive production performance of tobacco leaves:

1. The selected male sterile hybrid female parent, maintainer line and hybrid male parent are varieties with low nicotine conversion rate or purified low nicotine conversion rate varieties, and the conversion rate of the hybrid F1 is greatly reduced.

2. The nornicotine and nicotine conversion rate of the hybrid F1 had negative hybrid vigor, and when the nicotine conversion rate of the female parent was lower than that of the male parent, the negative heterosis of the hybrid F1 was greater.

3. Since the hybrid F1 seeds used are directly used in tobacco leaf production, the genotype is in a highly heterozygous state, so there is no need to worry about the weakening and loss of heterosis caused by gene segregation.

4. The nicotine conversion rate and nornicotine content of hybrid F1 decreased significantly, thereby reducing the content of tobacco-specific nitrosamines (TSNA) in tobacco leaves, especially the content of NNN (N-nitrosonornicotine), The problem of high TSNA content in tobacco leaf production can be solved fundamentally.

5. Combining with multiple breeding objectives such as "high quality, suitable yield, and disease resistance", the safety and comprehensive production performance of tobacco leaves are improved.

6. In addition to increasing the cost of sampling and testing for nicotine conversion, other procedures and methods can be the same as those of conventional varieties for purification and compounding, male sterile hybrid generation advantage utilization breeding methods, without increasing costs.

7. After the popularization and application of the new varieties selected and bred by the present invention, it is not necessary to further select and improve the conversion rate of nicotine.

Description of drawings

Figure 1 is the distribution frequency of nicotine in parents and reciprocal cross F1 populations;

Figure 2 is the distribution frequency of nornicotine in the parental and reciprocal cross F1 populations;

Figure 3 shows the distribution frequency of nicotine conversion rate in parental and reciprocal cross F1 populations.

Detailed ways

Below in conjunction with embodiment the present invention is further described.

Example 1: Test of the influence of different hybridization methods on the conversion rate of nicotine.

1. Test process and method:

1. In 2010, it was carried out in Enshi Tobacco Breeding Base. In order to avoid the influence of genetic differences in multiple traits among different varieties, the fertile lines of the same variety with different nicotine conversion rates (completely developed pistils and stamens, capable of self-fertilization) were selected as fertile lines (the pistil and stamens are fully developed and can be self-fertilized) after artificial multi-generation selection (the selection process is simple) Hybrid parent material. That is, Burley tobacco B37 strain with low nicotine conversion rate (referred to as P1) and B37 strain with high nicotine conversion rate (referred to as P2), 40 plants were planted in the field, and samples were taken at the stage of tobacco plant clusters to detect nicotine conversion rate, and P1 tobacco was selected. The excellent individual plants with strong growth and normal development with alkali conversion rate <5% and P2 nicotine conversion rate >50% were used to prepare orthogonal combination (P1×P2)F1 and reverse cross combination (P2×P1)F1.

2. In 2011, a total of four materials of P1, P2, orthogonal (P1×P2) F1 and reverse cross (P2×P1) F1 were planted in the field of Enshi Tobacco Breeding Breeding Base, of which P1 and P2 were each planted with 60 plants, and the forward and reverse 30 plants were planted in cross-cross F1. The planting method is the same as the large-scale production of Burley tobacco.

3. At the stage of clustering tobacco plants (tobacco plants are 30-40cm high and the number of leaves is 12-15 pieces), 55 plants are sampled in P1 and P2, and 20 plants are sampled in direct and reverse cross F1, and the 9th to 10th leaf position of the sampled plants is picked (From bottom to top) 2 pieces of tobacco leaves, after stringing, spray 0.3% ethephon solution to induce nicotine conversion rate, keep it in the room for 7-8 days, dry quickly after the tobacco leaves turn yellow, and make powder samples for use for the detection of alkaloid content.

4. Determination of alkaloids: Weigh 100 mg of each sample, and extract alkaloids with chloroform. Detection by gas chromatography. The gas chromatograph is Agilent-6890, the detector is FID, and the specific operation and parameter setting are carried out according to the method of Burton et al.

The nicotine conversion ability is represented by the nicotine conversion rate, and the calculation formula is: nicotine conversion rate (%)=[nornicotine content/(nicotine content+nornicotine content)]×100. According to the nicotine conversion rate, the tobacco strains are divided into non-converted strains (nicotine conversion rate is less than 5%), low-transformed strains (nicotine conversion rate 5%-20%), and medium-transformed strains (nicotine conversion rate 20%-20%). 50%) and high transforming strains (nicotine conversion rate greater than 50%).

5. Analysis of heterosis: the percentage of the hybrid F1 exceeding the average value (MP) of the parents. Calculation formula: heterosis (%)=[(F1-MP)/MP]×100.

2. Comparison of results

1. Comparison of population alkaloid content and nicotine conversion rate

It can be seen from Table 1 that there are large differences in the nicotine, nornicotine content and nicotine conversion rate of P1 and P2, among which the nicotine content of P1 (B37 low-nicotine conversion strain) is higher at 17.08-24.92mg/g , The content of nornicotine and the conversion rate of nicotine are low, which are 1.02-1.55mg/g and 4.09-6.68%, respectively; The content and conversion rate of nicotine are relatively high, ranging from 6.66 to 15.55mg/g and 41.51 to 87.56%, respectively. Orthogonal F1 nicotine content is higher at 14.36-19.08mg/g, nornicotine content and nicotine conversion rate are lower, respectively at 1.79-3.45mg/g and 9.33-16.80%; reverse-crossing F1 nicotine content is 12.11 ～16.73mg/g, nornicotine content and nicotine conversion rate are 3.35～5.20mg/g and 17.00～29.08% respectively; Between P1 and P2, but they are all biased towards P1, and the average nicotine content of the orthogonal F1 is higher than that of the reverse cross F1, while the nornicotine and nicotine conversion rate are lower than the reverse cross F1 and the orthogonal F1. See Table 2 for details.

Table 1 P1, P2 alkaloid content and nicotine conversion rate comparison mg/g, %

Table 2 Comparison of alkaloid content and nicotine conversion rate of forward and reverse cross F1 mg/g, %

2. Distribution frequency analysis

Analyze the distribution frequency of nicotine, nornicotine content and nicotine conversion rate of the tobacco plant population, the results are as shown in Figure 1-3 (Figure 1 is the distribution frequency of nicotine in the parents and reciprocal cross F1 population; Figure 2 is the distribution frequency of the parents and Distribution frequency of nornicotine in reciprocal cross F1 population; Figure 3 shows the distribution frequency of nicotine conversion rate in parents and reciprocal cross F1 population): nicotine content P1 frequency peak at 23mg/g, P2 at 7mg/g; F1 is at the 19mg/g site, which is 4mg/g less than P1 and 12mg/g more than P2; the reverse cross F1 is at 15mg/g site, which is less than P1 and 8mg/g more than P2. For nornicotine, the frequency peak of P1 is at 2 mg/g, and that of P2 is at 11 mg/g; the orthogonal F1 is at the 3 mg/g site, which is 1 mg/g higher than P1 and 8 mg/g lower than P2; the reverse crossing F1 is at 5 mg/g Site, 3mg/g more than P1, 6mg/g less than P2. The frequency peak of nicotine conversion rate of P1 is at 4%, and that of P2 is at 70%. Orthogonal F1 is at 15%, which is 11% higher than P1 and 55% lower than P2; P1 increased by 21 percentage points and decreased by 45 percentage points compared with P2. In short, the frequency peaks of nicotine, nornicotine and nicotine conversion rate of orthogonal F1 and reverse cross F1 are all between P1 and P2, but they are all shifted to P1, and the frequency peaks of orthogonal F1 are more obvious.

3. Analysis of heterosis

On the basis of the aforementioned average value comparison and distribution frequency analysis, the heterosis of reciprocal cross F1 was further analyzed, and the results showed that there was a positive mean nicotine content in both reciprocal and reciprocal F1, and the orthogonal F1 was greater than the reciprocal F1. For cross F1, they were 24.58% and 5.12%, respectively; nornicotine showed negative dominance, and orthogonal F1 was greater than reverse cross F1, which were -49.79% and -26.32%, respectively; the negative dominance of nicotine conversion rate was more obvious , Orthogonal F1 and Reversed F1 are -57.43% and -32.86%, respectively.

Table 3 Reciprocal cross heterosis analysis mg/g, %

(iii) Conclusion

1. From the numerical range of nicotine, nornicotine and nicotine conversion rate of the tobacco plant population, single plant distribution frequency, average value and heterosis analysis, whether it is a low nicotine conversion rate line and a high nicotine conversion rate hybrid ( Orthogonal), or high nicotine conversion rate strains crossed with low nicotine conversion rate strains (reverse cross), can obtain positive heterosis with nicotine content higher than the average value of the parents, nornicotine and nicotine conversion rate significantly Lower than the average value of the parents, with negative heterosis.

2. Using the line with low nicotine conversion rate as the female parent, the hybrid F1 (orthogonal) has greater heterosis.

Example 2: Comparative test on the superiority of male sterile hybrid F1 nicotine conversion rate.

1. Test process and method:

1. Example (1) is the result of crossing with fertile lines. This example uses male sterility (MS) crossing to verify the conclusion that it can produce negative heterosis for nicotine conversion rate through crossing.

2. In 2012, it was carried out in Enshi Tobacco Breeding Base. In order to exclude the influence of genetic differences of multiple traits in different varieties, different nicotine conversion rate types of the same variety were still selected as parents.

3. The test materials include 2 male sterile lines of Burley tobacco with low nicotine conversion rate, MSTN90-20 and MSB37-4, 2 parts of Burley tobacco with high nicotine conversion rate TN90-9, B37-46, and 1 part Burley tobacco male sterile line MSTN86-22 with high nicotine conversion rate, 1 part of TN86-12 with low nicotine conversion rate, 1 part of Maryland tobacco male sterile line MSMd609-4 with high nicotine conversion rate, 1 part of Maryland tobacco Tobacco line Md609-46 with low nicotine conversion rate. A total of 8 copies were processed. The selection process of low and high nicotine conversion rate strains is omitted.

40 plants were planted in the field. After testing the conversion rate of alkaloids and nicotine, selected excellent individual plants with normal development, and prepared 4 hybrid combinations respectively, which were 2 MS low conversion rate × high conversion combinations: hybrid combination 1 (MSNT -20×CT-9), hybrid combination 2 (MSB37-4×B37-46); 2 copies of MS high conversion rate×low conversion combination: hybrid combination 3 (MSTN86-22×TN86-12), hybrid combination 4 (MSMd609 -4 x Md609-46).

4. Combinations of 8 parents and 4 hybrid F1 in the planting step (3) in Enshi Tobacco Breeding Breeding Base in 2013. 60 plants were planted for each parent, and 50 plants were sampled, and 30 plants were planted for each hybrid F1, and 20 plants were sampled. The field planting method is the same as the large-scale production of Burley tobacco.

5. At the stage of clustering tobacco plants (tobacco plants are 30-40cm high, and the number of leaves is 12-15 pieces), take 2 pieces of growing and shaped tobacco leaves at the 9th-10th leaf position from bottom to top, and spray 0.3% ethylene after stringing Tobacco solution was used to induce the conversion rate of nicotine, and it was dried in the air for 7-8 days with moisture in the room. After the tobacco leaves turned yellow, they were quickly dried to make powder samples.

6. Determination of nicotine and nornicotine: Weigh 100 mg of each sample, and extract alkaloids with chloroform. Detection by gas chromatography. The gas chromatograph is Agilent-6890, the detector is FID, and the specific operation and parameter setting are carried out according to the method of Burton et al.

The nicotine conversion ability is represented by the nicotine conversion rate, and the calculation formula is: nicotine conversion rate (%)=[nornicotine content/(nicotine content+nornicotine content)]×100. According to the nicotine conversion rate, the tobacco strains are divided into non-converted strains (nicotine conversion rate is less than 5%), low-transformed strains (nicotine conversion rate 5%-20%), and medium-transformed strains (nicotine conversion rate 20%-20%). 50%) and high transforming strains (nicotine conversion rate greater than 50%).

7. Analysis of heterosis: Heterosis refers to the phenomenon that certain traits of the first generation of hybrids are superior to those of their parents. That is: the percentage of the first generation of hybrids (F1) exceeding the mean (MP) of the parents. Calculation formula: heterosis (%)=[(F1-MP)/MP]×100.

2. Comparison of results

1. Comparison of heterosis in nicotine conversion rate of male sterile low-transformation line × high-transformation line F1

The male sterile line with low nicotine conversion rate was used as the female parent, and the fertile line with high nicotine conversion rate was used as the male parent. Hybrid combination 1 (MSNT-20×TN-90) F1 and hybrid combination 2 (MSB37- 4×B37-46)F1, the heterosis of nicotine content were all positive, being 20.16% and 18.87%, respectively, and the heterosis of nornicotine content were all negative, being -45.69% and -42.46%, respectively. The two hybrid combinations are -50.51% and -46.58%, respectively. From the calculation formulas of nicotine conversion rate and heterosis, it is not difficult to know that due to the positive dominance of nicotine content and the negative dominance of nornicotine, and nornicotine The advantage of content is greater than that of nicotine, which leads to the generation of negative advantage of nicotine conversion rate.

Table 4 Heterosis analysis of nicotine conversion rate in 2 male sterile low transformation lines×high transformation lines F1

2. Comparison of heterosis in nicotine conversion rate of male sterile high-transformation line×low-transformation line F1

It can be seen from Table 5 that the male sterile line with high nicotine conversion rate was used as the female parent, and the fertile line with low nicotine conversion rate was used as the male parent, and the Burley tobacco hybrid combination 3 (MSTN86-22×TN86-12 ) and Maryland tobacco hybrid combination 4 (MSMd609-14×Md609-8), the nicotine content also showed positive heterosis, but the heterosis was quite small, which was close to the mean value (MP) of the parents, which were 4.46% and 1.85%, respectively. Nornicotine was negatively dominant, respectively -12.57% and -12.81%, and the conversion rate of nicotine was also negatively dominant, respectively -13.9% and -10.91%.

Table 5 Heterosis analysis of nicotine conversion rate in 2 male sterile high transformation lines×low transformation lines F1

(iii) Conclusion

1. The male sterile line of Burley tobacco and Maryland tobacco was used as the female parent, and the corresponding fertile line material was used as the male parent. According to the level of nicotine conversion rate, MS high × low and MS low × high were prepared respectively. 4 hybrid F1 combinations. The results showed that both MS high × low combination and MS low × high combination could obtain positive heterosis for nicotine content and negative heterosis for nornicotine and nicotine conversion rate.

2. The positive advantages of nicotine content and the negative advantages of nornicotine and nicotine conversion rate of MS low × high combination are significantly higher than those of MS high × low.

3. The test result is consistent with the conclusion of Example 1.

Example 3, the comparison of the effects of reducing tobacco-specific nitrosamines (TSNA) in male sterile hybrid F1

1. Test steps and methods

1. Select the male sterility cross generation advantage utilization of tobacco as the breeding method.

2. From 1998 to 2011, the multi-generation nicotine conversion rate selection and purification were carried out on the male sterile lines MSB21, MSTN86 and the corresponding maintainer lines B21, TN86, B37, and LAB21 respectively (purification process omitted). In 2012, after comparative identification, 6 copies The nicotine conversion rates of the varieties are all less than 5%, and the nicotine conversion rates and other characters of the sterile line and maintainer line are completely consistent except for fertility. "LC" (English abbreviation for low nicotine conversion) is added after the name of the purified variety to show the difference from the original variety.

3. According to step (2) in 2013, Burley tobacco MSB21LC and MSTN86LC after multiple generations of purification were selected as female parents, and B37LC and LAB21LC were used as male parents. In the field, 80 plants were planted for each female parent, and 0 plants were planted for each male parent. According to the routine Burley Tobacco Seed Propagation Technology Standardizes Cultivation and Planting Management.

4. Conduct early induction of nicotine conversion rate and detection of alkaloids on the father and mother parents in the step (3) at the tobacco plant group stage. That is, when the tobacco plant is 30-40cm high and the number of leaves is 12-15, collect 1-2 leaves at the 8th-10th leaf position from bottom to top according to the number of each plant, and spray 0.3% ethylene on the leaves after stringing. Carry out the nicotine conversion rate induction treatment, keep it in the air for 7-8 days, and after the leaves turn yellow completely, dry them according to the individual plant number to make powder samples, detect the content of nicotine and nornicotine, and calculate the nicotine conversion Rate. Calculation formula: nicotine conversion rate (%)=[nornicotine/(nicotine+nornicotine)]×100. Nicotine conversion rate <5% is non-conversion, 5-20% is low conversion, 20-50% is medium conversion, and >50% is high conversion. According to the detection results of the transformation rate, find the corresponding number of the individual plant in the field, and eliminate the transformed strains with a nicotine conversion rate > 5%. Among them, the nicotine conversion rate of the female parent was more than 3%.

5. Planting the parents retained in the step (4), performing artificial cross pollination, and harvesting hybrid seeds. Namely (MSB21LC×B37LC)F1 and MSTN86LC×LAB21LC)F1.

6. In 2014, a comparative test was carried out on the hybrid seeds (MSB21LC×B37LC)F1 and (MSTN86LC×LAB21LC)F1 harvested in step (5). The unselected Eyan 1 (MSB21×B37) F1 and Eyan 3 (MSTN86×LAB21) F1 were used as controls. A total of 4 materials were used for comparative experiments in large plots, with three repetitions, four rows of plots, 40 plants per plot. Conventional burley tobacco production technology cultivation and modulation technology.

7. After the tobacco leaf preparation in step (6) is finished, remove 2 pieces of the lower part (7-8 leaf positions from bottom to top), 2 pieces of the middle part (13-14 leaf positions), and 2 pieces of the upper part (18 ~19 leaf positions), and mixed samples were made according to the parts to detect alkaloids, nicotine conversion rate, and tobacco-specific nitrosamines (TSNA).

8. Specific methods for detecting alkaloids, nicotine conversion rates, and tobacco-specific nitrosamines (TSNA) in step (7):

The detection of alkaloids adopts the gas chromatography method introduced by Lian Yunyun et al. Agilent 7890A gas chromatograph and FID detector were used to measure the content of nicotine and nornicotine, and the conversion rate of nicotine was calculated according to the measurement results. The formula:

Nicotine conversion rate = [nornicotine content / (nicotine content + nornicotine content)] × 100%

For the detection of TSNA, according to the ammonium acetate extraction-LC-MS method introduced by Ding Shichao et al., Agilent 6460A triple quadrupole liquid-mass spectrometer was used to determine the contents of four kinds of TSNA.

9. According to the test results in step (8), calculate the control advantage, and compare (MSTN86LC×LAB21LC)F1 and the corresponding control in reducing nicotine conversion rate and TSNA effect. Control Dominance (%)=[(F1-Control)/Control]×100.

(ii) Results

1. Detection results of alkaloid and nicotine conversion rate

(MSB21LC×B37LC) The nicotine content of the three parts of F1 was 27.01-54.40mg/g, and the control advantage was 7.81-12.00%. The nornicotine content was 1.23-2.33mg/g, and the control advantage was negative, at -83.05- Between -86.57%; the conversion rate of nicotine is 3.58-4.36%, and the comparative advantage is negative, at -82.58--83.92%.

(MSTN86LC×LAB21LC) The content of nicotine in the three parts of F1 is 22.07～42.08mg/g, the control advantage is 3.03～5.12%; the content of nornicotine is 0.86～1.64mg/g, the control advantage is negative, at -71.97～- 79.43%; the conversion rate of nicotine is 3.61-3.75%, and the control advantage is negative, ranging from -70.29 to -77.03%.

Table 6 Comparison of conversion rates of alkaloids and nicotine mg/g, %

2. TSNA test results

(MSB21LC×B37LC) The content of NNN in the three parts of F1 is 17.59～26.00ug/g, the comparative advantage is negative, -64.18～-70.23%, the total amount of TSNA is 24.83～34.38ug/g, the comparative advantage is -57.46～- 62.56%.

(MSTN86LC×LAB21LC) The content of NNN in the three parts of F1 was 5.50～6.95ng/g, the comparative advantage was -71.84～-78.22%, the total amount of TSNA was 11.30～-13.55ug/g, the comparative advantage was -55.98～-69.62%.

Table 4 NNN content and TSNA total amount comparison ug/g, %

(iii) Conclusion

On the basis of examples (1) and (2) proving that the negative heterosis of F1 nicotine conversion can be obtained through the utilization of heterosis, and the negative heterosis of F1 is greater with the low-transformation line as the female parent. Multi-generation selection of purified low nicotine to transform the parent and female parents, the prepared male sterile hybrid generation (i.e. low × low), can greatly reduce nornicotine content, nicotine conversion rate, NNN content and the total amount of TSNA , compared with the control, the effect of reducing the conversion rate of nicotine is over 70%, the effect of reducing NNN is over 60%, and the total amount of TSNA is over 50%.

Claims (2)

1. one kind is reduced the breeding method of tobacco smoke alkaloid conversion ratio, it is characterized in that, adopt male sterile first generation of hybrid use of advantage method, select the maternal and maintainer of male sterile and male parent, the nicotine conversion rate of male sterile line hybridization maternal and maintainer and male parent must lower than 5%, and male sterile line hybridization nicotine conversion rate that is maternal and maintainer must be less than male parent; Nicotine conversion rate higher than 5% the maternal and maintainer of male sterile line hybridization and male parent must carry out nicotine conversion rate and select and purify; Nicotine conversion rate is hybridized maternal nicotine conversion rate select to comprise male sterile line female parent with purification and maintainer is synchronously purified higher than the male sterile line of 5%;

Concrete breeding step is as follows:

1), the maternal and maintainer of male sterile line and parent sire of hybrid pigs respectively plant 40-60 strain, and leaf tobacco production mode is applied fertilizer, cultivated routinely;

2), during, number of blade 12-15 sheet high at cigarette strain 30-40cm, number by individual plant, gather the growth qualitative blade 1-2 sheet of 8-10 leaf position from bottom to top, after string rope, the ethrel of foliage-spray 0.3% carries out nicotine conversion rate induction process, to dry in the air 7-8d processed in indoor moisturizing, after blade turns yellow completely, dry respectively by individual plant numbering and make powder sample, detect nicotine and nornicotine content, calculate nicotine conversion rate;

3), according to conversion ratio testing result, find field individual plant reference numeral, retain the individual plant that 5-10 strain conversion ratio is quite lower, pinch or extract other higher transformant superseded;

4), in maintainer and male parent colony in the individual plant of choosing carry out bagging selfing and reserve seed for planting, after seed collection, mixing gathers; The non-transformed strain hybridization pollination in the pollen of maintainer individual plant and male sterile line colony is selected in collection;

5), after the seed maturity of middle menu strain, gather by individual plant number, after threshing, press Cultivar mixtures, indicate variety name, select from generation to generation for next year.

2. the method for claim 1, it is characterized in that, nicotine conversion rate is selected need repeat 3 ~ 4 generations more than with purification, until male sterile line, maintainer and male parent colony plant are the non-transformed strain of conversion ratio < 5% completely, male sterile line and maintainer are except fertility, nicotine conversion rate is completely the same with other proterties, can combine by preparing hybrid F1.